Long-range DNA end resection supports homologous recombination by checkpoint activation rather than extensive homology generation
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Curated by eLife
eLife assessment:
This fundamental study provides compelling evidence that long-range resection is important for recombination between distal, but not proximal, homologous sequences. It is thus proposed that a major role of long resection of a double-strand break mediated by Sgs1 and Exo1 is to activate the DNA damage checkpoint to allow the chromosomal mobility needed for the DNA ends to find a distant homologous sequence with which repair via homologous recombination, adding a new biological meaning to the role of long DNA resection in homologous recombination.
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Abstract
Homologous recombination (HR), the high-fidelity mechanism for double-strand break (DSB) repair, relies on DNA end resection by nucleolytic degradation of the 5′-terminated ends. However, the role of long-range resection mediated by Exo1 and/or Sgs1-Dna2 in HR is not fully understood. Here, we show that Exo1 and Sgs1 are dispensable for recombination between closely linked repeats, but are required for interchromosomal repeat recombination in Saccharomyces cerevisiae . This context-specific requirement for long-range end resection is connected to its role in activating the DNA damage checkpoint. Consistent with this role, checkpoint mutants also show a defect specifically in interchromosomal recombination. Furthermore, artificial activation of the checkpoint partially restores interchromosomal recombination to exo1∆ sgs1∆ cells. However, cell cycle delay is insufficient to rescue the interchromosomal recombination defect of exo1∆ sgs1∆ cells, suggesting an additional role for the checkpoint. Given that the checkpoint is necessary for DNA damage-induced chromosome mobility, we propose that the importance of the checkpoint, and therefore long-range resection, in interchromosomal recombination is due to a need to increase chromosome mobility to facilitate pairing of distant sites. The need for long-range resection is circumvented when the DSB and its repair template are in close proximity.
Article activity feed
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eLife assessment:
This fundamental study provides compelling evidence that long-range resection is important for recombination between distal, but not proximal, homologous sequences. It is thus proposed that a major role of long resection of a double-strand break mediated by Sgs1 and Exo1 is to activate the DNA damage checkpoint to allow the chromosomal mobility needed for the DNA ends to find a distant homologous sequence with which repair via homologous recombination, adding a new biological meaning to the role of long DNA resection in homologous recombination.
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Reviewer #1 (Public Review):
This is a nice and elegant genetic study on the role of the Sgs1 and Exo1 factors involved in long DNA resection in the mechanism of double-strand break (DSB) repair by homologous recombination (HR). Most studies have focused on the need for these two factors for the long resection of a DSB to allow efficient HR. Now, this study shows that a major role of the function of long resection mediated by Sgs1 and Exo1 is to activate the DNA damage checkpoint to allow the chromosomal mobility needed to allow the DNA ends to find a distant homologous sequence with which repair via homologous recombination.
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Reviewer #2 (Public Review):
A new study by Kimble et al. examines the role of extensive resection in DNA double-strand break repair. Formation of ssDNA at DNA breaks is initiated by Mre11-Rad50-Xrs2 and followed by Exo1 or Sgs1/Dna2, which form longer ssDNA. This ssDNA is used to load recombination and DNA damage checkpoint proteins. Some studies suggested that very short ssDNA by MRX complex is sufficient for DSB repair. Here, the authors look carefully at the role of extensive resection in DSB repair by gene conversion. To address this question they have constructed a large number of new recombination assays. They find that sgs1 exo1 mutants that lack extensive resection are capable of DSB repair when recombining loci are present on a single DNA molecule and within 50 kb from each other. When the template for DSB repair is further …
Reviewer #2 (Public Review):
A new study by Kimble et al. examines the role of extensive resection in DNA double-strand break repair. Formation of ssDNA at DNA breaks is initiated by Mre11-Rad50-Xrs2 and followed by Exo1 or Sgs1/Dna2, which form longer ssDNA. This ssDNA is used to load recombination and DNA damage checkpoint proteins. Some studies suggested that very short ssDNA by MRX complex is sufficient for DSB repair. Here, the authors look carefully at the role of extensive resection in DSB repair by gene conversion. To address this question they have constructed a large number of new recombination assays. They find that sgs1 exo1 mutants that lack extensive resection are capable of DSB repair when recombining loci are present on a single DNA molecule and within 50 kb from each other. When the template for DSB repair is further away on the same molecule or present on a different chromosome, the repair is reduced by 5-10 folds in the absence of extensive resection. The authors present data suggesting that this defect relates to slower repair kinetics between more distant homologous sequences and the need for a Mec1-mediated DNA damage checkpoint that requires extensive resection. The role of the checkpoint response is likely not limited to simple cell cycle arrest but may also be necessary for the mobility of a broken molecule. Partial suppression of the sgs1 exo1 repair defect is accomplished by activating the checkpoint using an artificial system colocalizing checkpoint proteins on a separate chromosome. Altogether the manuscript addresses an important question, is well-written, and presents interesting data.
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Reviewer #3 (Public Review):
This manuscript aims to define the importance of long-range resection for homologous recombination, a relevant and yet unanswered question in the field of genome maintenance. The data shows that long-range resection is required for interchromosomal, but not intrachromosomal, recombination is well-developed and convincing. The claim that the DNA damage checkpoint is crucial for promoting distal recombination is interesting and founded on logical rationale. However, some key points about the proposed role of checkpoint signaling and the presented results need further clarification, mainly regarding the issue of checkpoint activation status in exo1Δ sgs1Δ cells and the attempts of using forced Rad53 activation to rescue interchromosomal recombination defects. Additional experiments would help solidify the …
Reviewer #3 (Public Review):
This manuscript aims to define the importance of long-range resection for homologous recombination, a relevant and yet unanswered question in the field of genome maintenance. The data shows that long-range resection is required for interchromosomal, but not intrachromosomal, recombination is well-developed and convincing. The claim that the DNA damage checkpoint is crucial for promoting distal recombination is interesting and founded on logical rationale. However, some key points about the proposed role of checkpoint signaling and the presented results need further clarification, mainly regarding the issue of checkpoint activation status in exo1Δ sgs1Δ cells and the attempts of using forced Rad53 activation to rescue interchromosomal recombination defects. Additional experiments would help solidify the proposed model. Nonetheless, the paper establishes the importance of long-range resection for distal recombination and should be considered a significant contribution to the field.
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